Direct visualization of receptor arrays in frozen‐hydrated sections and plunge‐frozen specimens of <i>E. coli</i> engineered to overproduce the chemotaxis receptor Tsr

524

472

The cell walls of mycobacteria form an exceptional permeability barrier, and they are essential for virulence. They contain extractable lipids and long-chain mycolic acids that are covalently linked to peptidoglycan via an arabinogalactan network. The lipids were thought to form an asymmetrical bilayer of considerable thickness, but this could never be proven directly by microscopy or other means. Cryo-electron tomography of unperturbed or detergenttreated cells of Mycobacterium smegmatis embedded in vitreous ice now reveals the native organization of the cell envelope and its delineation into several distinct layers. The 3D data and the investigation of ultrathin frozen-hydrated cryosections of M. smegmatis, Myobacterium bovis bacillus Calmette-Gué rin, and Corynebacterium glutamicum identified the outermost layer as a morphologically symmetrical lipid bilayer. The structure of the mycobacterial outer membrane necessitates considerable revision of the current view of its architecture. Conceivable models are proposed and discussed. These results are crucial for the investigation and understanding of transport processes across the mycobacterial cell wall, and they are of particular medical relevance in the case of pathogenic mycobacteria.bacterial cell wall ͉ Corynebacterium glutamicum ͉ Mycobacterium bovis ͉ Mycobacterium smegmatis ͉ mycolic acid layer M ycobacteria have evolved a complex cell wall, comprising a peptidoglycan-arabinogalactan polymer with covalently bound mycolic acids of considerable size (up to 90 carbon atoms), a variety of extractable lipids, and pore-forming proteins (1-3). The cell wall provides an extraordinarily efficient permeability barrier to noxious compounds and contributes to the high intrinsic resistance of mycobacteria to many drugs (4). Because of the paramount medical importance of Mycobacterium tuberculosis, the ultrastructure of mycobacterial cell envelopes has been intensively studied during recent decades. The current view of the cell wall architecture is essentially based on a model suggested by Minnikin (5). He proposed that the covalently bound mycolic acids form the inner leaflet of an asymmetrical bilayer. Other lipids extractable by organic solvents were thought to form the outer leaflet, either intercalating with the mycolates (5, 6) or forming a more clearly defined interlayer plane (7). Elegant x-ray diffraction studies proved that the mycolic acids are oriented parallel to each other and perpendicular to the plane of the cell envelope (8). Furthermore, freeze-fracture studies showed a second fracture plane in electron micrographs (9), indicating the existence of a hydrophobic bilayer structure external to that of the cytoplasmic membrane. Mutants or treatments affecting mycolic acid biosynthesis and the production of extractable lipids resulted in an increase of cell wall permeability in various mycobacteria and related microorganisms (10-12) and a drastic decrease of virulence, underlining the importance of the integrity of the cell wall for intracellular survival...

Section: Discussionmentioning

confidence: 63%

Disclosure of the mycobacterial outer membrane: Cryo-electron tomography and vitreous sections reveal the lipid bilayer structure

Hoffmann

Leis

Niederweis

et al. 2008

524

472

“…We have found that immersion of a hippocampal organotypic brain slice in ACSF supplemented with 5% sucrose and 20% dextran for 5 min before freezing is the minimum requirement to achieve reproducible vitrification. Note that 20% dextran is routinely used for extracellular cryoprotection of cell suspension because it produces minimal osmotic effect (17)(18)(19).…”

Section: Establishment Of the Best Freezing Conditionsmentioning

confidence: 99%

“…Structures are preserved down to atomic resolution, and the observed contrast is directly related to density variations in the sample. The method, however, only works for particles or cells that can be squeezed in a sub-micrometer-thick vitreous film.Cryo-electron microscopy of vitreous section (CEMOVIS) was developed to extend the advantage of vitrification to any cell and tissue (7,(16)(17)(18)(19)(20)(21). In general, high-pressure cooling is used to vitrify samples of several hundred-micrometer thickness with minimal addition of cryoprotectant (22).…”

mentioning

confidence: 99%

The mammalian central nervous synaptic cleft contains a high density of periodically organized complexes

Zuber

Nikonenko

Klauser

et al. 2005

206

176

Cryo-electron microscopy of vitreous section makes it possible to observe cells and tissues at high resolution in a close-to-native state. The specimen remains hydrated; chemical fixation and staining are fully avoided. There is minimal molecular aggregation and the density observed in the image corresponds to the density in the object. Accordingly, organotypic hippocampal rat slices were vitrified under high pressure and controlled cryoprotection conditions, cryosectioned at a final thickness of Ϸ70 nm and observed below ؊170°C in a transmission electron microscope. The general aspect of the tissue compares with previous electron microscopy observations. The detailed analysis of the synapse reveals that the density of material in the synaptic cleft is high, even higher than in the cytoplasm, and that it is organized in 8.2-nm periodic transcleft complexes. Previously undescribed structures of presynaptic and postsynaptic elements are also described.Cryo-electron microscopy of vitreous section ͉ high-pressure freezing ͉ hippocampus S ynapses of the central nervous system (CNS) play a key role in neuronal information processing. Their physiology and structural organization have been extensively characterized (1, 2). The presynaptic compartment contains synaptic vesicles (SVs) filled with neurotransmitters. There is an intensive tethering and fusion activity between SVs and the presynaptic membrane. The postsynaptic membrane is covered with neurotransmitter receptors, which detect variations in neurotransmitter concentration. Postsynaptic submembrane cytoplasm is occupied by a complex network of proteins, the postsynaptic density, which modulates the strength of synaptic transmission. The space between both cells is the synaptic cleft. It contains neurotransmitter receptors and various adhesion molecules, such as N-cadherin and neural cell adhesion molecule. Their function in synapse formation and regulation is only beginning to be understood, and their precise spatial organization remains unclear (3).Resolving the molecular structure of the synapse is difficult because, on one hand, high resolution x-ray diffraction data are obtained outside their cellular context (4, 5). On the other hand, conventional electron microscopy generally fails to resolve individual proteins in their natural environment because the preparation relies on chemical fixation, dehydration, resinembedding, and heavy-metal staining and, thus, produces aggregation artifacts and artificial contrast, resulting in a limited resolution and a difficulty of interpreting the nature of the observed structures (6, 7). Freeze substitution and freeze etching, by which the specimen is immobilized by freezing and dehydrated at low temperature before being embedded in resin or replicated, were developed to reduce preparation artifacts but, even under these conditions, aggregation cannot be completely avoided. Depending on the protocols used for specimen preparation, the synaptic cleft presents different aspects. A dense central plaque was observed in some s...

“…It was further proposed that, in E. coli, trimers of receptor dimers form a hexagonal array with a lattice spacing of 20 nm (13). A subsequent electron cryotomography (ECT) study showed that overexpressed Tsr chemoreceptors in E. coli pack into a hexagonal lattice with a center-to-center spacing of 7.5 nm (14)(15)(16)(17). In these overexpression strains, the receptors surprisingly form a ''zipper-like'' double layer, in which large invaginations of the inner membrane allow the cytoplasmic tips of one layer to interact with the cytoplasmic tips of a second, facing layer.…”

mentioning

confidence: 99%

Universal architecture of bacterial chemoreceptor arrays

Briegel

Ortega

Tocheva³

et al. 2009

295

405

Chemoreceptors are key components of the high-performance signal transduction system that controls bacterial chemotaxis. Chemoreceptors are typically localized in a cluster at the cell pole, where interactions among the receptors in the cluster are thought to contribute to the high sensitivity, wide dynamic range, and precise adaptation of the signaling system. Previous structural and genomic studies have produced conflicting models, however, for the arrangement of the chemoreceptors in the clusters. Using whole-cell electron cryo-tomography, here we show that chemoreceptors of different classes and in many different species representing several major bacterial phyla are all arranged into a highly conserved, 12-nm hexagonal array consistent with the proposed ''trimer of dimers'' organization. The various observed lengths of the receptors confirm current models for the methylation, flexible bundle, signaling, and linker sub-domains in vivo. Our results suggest that the basic mechanism and function of receptor clustering is universal among bacterial species and was thus conserved during evolution.bacterial ultrastructure ͉ chemotaxis ͉ electron cryo-tomography